CTLA4-Ig and anti-CD40 ligand prevent renal allograft rejection in primates

Selective inhibition of T cell costimulation using the B7-specific fusion protein CTLA4-Ig has been shown to induce long-term allograft survival in rodents. Antibodies preventing the interaction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5–8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20–98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.

Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases

The 2.15-Å resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3′ to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3′ phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

Cerebral cortical astroglia from the trisomy 16 mouse, a model for Down syndrome, produce neuronal cholinergic deficits in cell culture

Trisomy 21 (Down syndrome) is associated with a high incidence of Alzheimer disease and with deficits in cholinergic function in humans. We used the trisomy 16 (Ts16) mouse model for Down syndrome to identify the cellular basis for the cholinergic dysfunction. Cholinergic neurons and cerebral cortical astroglia, obtained separately from Ts16 mouse fetuses and their euploid littermates, were cultured in various combinations. Choline acetyltransferase activity and cholinergic neuron number were both depressed in cultures in which both neurons and glia were derived from Ts16 fetuses. Cholinergic function of normal neurons was significantly down-regulated by coculture with Ts16 glia. Conversely, neurons from Ts16 animals could express normal cholinergic function when grown with normal glia. These observations indicate that astroglia may contribute strongly to the abnormal cholinergic function in the mouse Ts16 model for Down syndrome. The Ts16 glia could lack a cholinergic supporting factor present in normal glia or contain a factor that down-regulates cholinergic function.

Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells

Contact of cultured mammary epithelial cells with the basement membrane protein laminin induces multiple responses, including cell shape changes, growth arrest, and, in the presence of prolactin, transcription of the milk protein β-casein. We sought to identify the specific laminin receptor(s) mediating the multiple cell responses to laminin. Using assays with clonal mammary epithelial cells, we reveal distinct functions for the α6β4 integrin, β1 integrins, and an E3 laminin receptor. Signals from laminin for β-casein expression were inhibited in the presence of function-blocking antibodies against both the α6 and β1 integrin subunits and by the laminin E3 fragment. The α6-blocking antibody perturbed signals mediated by the α6β4 integrin, and the β1-blocking antibody perturbed signals mediated by another integrin, the α subunit(s) of which remains to be determined. Neither α6- nor β1-blocking antibodies perturbed the cell shape changes resulting from cell exposure to laminin. However, the E3 laminin fragment and heparin both inhibited cell shape changes induced by laminin, thereby implicating an E3 laminin receptor in this function. These results elucidate the multiplicity of cell-extracellular matrix interactions required to integrate cell structure and signaling and ultimately permit normal cell function.

Induction of HSP70 and polyubiquitin expression associated with plant virus replication

By examining the front of virus invasion in immature pea embryos infected with pea seed-borne mosaic virus (PSbMV), the selective control of different host genes has been observed. From our observations, the early responses to PSbMV replication can be grouped into three classes, inhibited host gene expression, induced host gene expression, and no effect on a normal host function. The expression of two heat-inducible genes encoding HSP70 and polyubiquitin was induced coordinately with the onset of virus replication and the down-regulation of two other genes encoding lipoxygenase and heat shock cognate protein. The down-regulation was part of a general suppression of host gene expression that may be achieved through the degradation of host transcripts. We discuss the possibilities of whether the induction of HSP70 and polyubiquitin genes represents a requirement for the respective protein products by the virus or is merely a consequence of the depletion of other host transcripts. The former is feasible, as the induction of both genes does result in increased HSP70 and ubiquitin accumulation. This also indicates that, in contrast to some animal virus infections, there is not a general inhibition of translation of host mRNAs following PSbMV infection. This selective control of host gene expression was observed in all cell types of the embryo and identifies mechanisms of cellular disruption that could act as triggers for symptom expression.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger AE1 have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in AE1. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in AE1 were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to AE1. In contrast, heterozygous mutations in AE1 were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a de novo mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in AE1 cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Adenoviral gene transfer of Caenorhabditis elegans n−3 fatty acid desaturase optimizes fatty acid composition in mammalian cells

Omega−3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n−3 PUFAs is insufficient in most Western people, whereas the level of n−6 PUFAs is relatively too high, with an n−6/n−3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n−3 PUFAs in mammals relies on chronic dietary intake of fat rich in n−3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n−3 PUFAs or to convert n−6 to n−3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n−3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n−3 PUFA contents and dramatically balance the ratio of n−6/n−3 PUFAs. Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n−6 PUFAs to the corresponding n−3 PUFAs, and changed the n−6/n−3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n−6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.

The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1/MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the alpha subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the beta subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AML1 (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression prevented tumor growth. These results suggest that expression of AML1/EAP and AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Simulation of the secondary electrons energy deposition produced by proton beams in PMMA: influence of the target electronic excitation description

We have studied the radial dependence of the energy deposition of the secondary electron generated by swift proton beams incident with energies T = 50 keV–5 MeV on poly(methylmethacrylate) (PMMA). Two different approaches have been used to model the electronic excitation spectrum of PMMA through its energy loss function (ELF), namely the extended-Drude ELF and the Mermin ELF. The singly differential cross section and the total cross section for ionization, as well as the average energy of the generated secondary electrons, show sizeable differences at T ⩽ 0.1 MeV when evaluated with these two ELF models. In order to know the radial distribution around the proton track of the energy deposited by the cascade of secondary electrons, a simulation has been performed that follows the motion of the electrons through the target taking into account both the inelastic interactions (via electronic ionizations and excitations as well as electron-phonon and electron trapping by polaron creation) and the elastic interactions. The radial distribution of the energy deposited by the secondary electrons around the proton track shows notable differences between the simulations performed with the extended-Drude ELF or the Mermin ELF, being the former more spread out (and, therefore, less peaked) than the latter. The highest intensity and sharpness of the deposited energy distributions takes place for proton beams incident with T ~ 0.1–1 MeV. We have also studied the influence in the radial distribution of deposited energy of using a full energy distribution of secondary electrons generated by proton impact or using a single value (namely, the average value of the distribution); our results show that differences between both simulations become important for proton energies larger than ~0.1 MeV. The results presented in this work have potential applications in materials science, as well as hadron therapy (due to the use of PMMA as a tissue phantom) in order to properly consider the generation of electrons by proton beams and their subsequent transport and energy deposition through the target in nanometric scales.

Evolução da tiroidite de hashimoto

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Le système endocannabinoïde dans la rétine du singe : expression, localisation et fonctions

Le cannabis produit de nombreux effets psychologiques et physiologiques sur le corps humain. Les molécules contenues dans cette plante, désignées comme « phytocannabinoïdes », activent un système endogène qu’on appelle le système endocannabinoïde (eCB). Les effets de la consommation de cannabis sur la vision ont déjà été décrits sans cependant de formulation sur les mécanismes sous-jacents. Ces résultats comportementaux suggèrent, malgré tout, la présence de ce système eCB dans le système visuel, et particulièrement dans la rétine. Cette thèse vise donc à caractériser l’expression, la localisation et le rôle du système eCB dans la rétine du singe vervet, une espèce animale ayant un système visuel semblable à celui de l’humain. Nous avons mis au point un protocole expérimental d’immunohistochimie décrit dans l’article apparaissant dans l’Annexe I que nous avons utilisé pour répondre à notre objectif principal. Dans une première série de quatre articles, nous avons ainsi caractérisé l’expression et la localisation de deux récepteurs eCBs reconnus, les récepteurs cannabinoïdes de type 1 (CB1R) et de type 2 (CB2R), et d’un 3e présumé récepteur aux cannabinoïdes, le récepteur GPR55. Dans l’article 1, nous avons démontré que CB1R et une enzyme clé de ce système, la fatty acid amide hydrolase (FAAH), sont exprimés dans les parties centrale et périphérique de la rétine, et abondamment présents dans la fovéa, une région où l’acuité visuelle est maximale. Dans l’article 2, nous avons localisé le CB2R dans des cellules gliales de la rétine : les cellules de Müller et nous avons proposé un modèle sur l’action de cette protéine dans la fonction rétinienne faisant appel à une cascade chimique impliquant les canaux potassiques. Dans l’article 3, nous avons observé le GPR55 exclusivement dans les bâtonnets qui sont responsables de la vision scotopique et nous avons soumis un deuxième modèle de fonctionnement de ce récepteur par le biais d'une modulation des canaux calciques et sodiques des bâtonnets. Vu que ces 3 récepteurs se retrouvent dans des cellules distinctes, nous avons suggéré leur rôle primordial dans l’analyse de l’information visuelle au niveau rétinien. Dans l’article 4, nous avons effectué une analyse comparative de l’expression du système eCB dans la rétine de souris, de toupayes (petits mammifères insectivores qui sont sont considérés comme l’étape intermédiaire entre les rongeurs et les primates) et de deux espèces de singe (le vervet et le rhésus). Ces résultats nous ont menés à présenter une hypothèse évolutionniste quant à l’apparition et à la fonction précise de ces récepteurs. Dans les articles subséquents, nous avons confirmé notre hypothèse sur le rôle spécifique de ces trois récepteurs par l’utilisation de l’électrorétinographie (ERG) après injection intravitréenne d’agonistes et d’antagonistes de ces récepteurs. Nous avons conclu sur leur influence indéniable dans le processus visuel rétinien chez le primate. Dans l’article 5, nous avons établi le protocole d’enregistrement ERG normalisé sur le singe vervet, et nous avons produit un atlas d’ondes ERG spécifique à cette espèce, selon les règles de l’International Society for Clinical Electrophysiology of Vision (ISCEV). Les patrons électrorétinographiques se sont avérés semblables à ceux de l’humain et ont confirmé la similarité entre ces deux espèces. Dans l’article 6, nous avons démontré que le blocage de CB1R ou CB2R entraine une modification de l’électrorétinogramme, tant au niveau photopique que scotopique, ce qui supporte l’implication de ces récepteurs dans la modulation des ondes de l’ERG. Finalement, dans l’article 7, nous avons confirmé le modèle neurochimique proposé dans l’article 3 pour expliquer le rôle fonctionnel de GPR55, en montrant que l’activation ou le blocage de ce récepteur, respectivement par un agoniste (lysophosphatidylglucoside, LPG) ou un antagoniste (CID16020046), entraine soit une augmentation ou une baisse significative de l’ERG scotopique seulement. Ces données, prises ensemble, démontrent que les récepteurs CB1R, CB2R et GPR55 sont exprimés dans des types cellulaires bien distincts de la rétine du singe et ont chacun un rôle spécifique. L’importance de notre travail se manifeste aussi par des applications cliniques en permettant le développement de cibles pharmacologiques potentielles dans le traitement des maladies de la rétine.

Higher order elicitability and Osband’s principle

A statistical functional, such as the mean or the median, is called elicitable if there is a scoring function or loss function such that the correct forecast of the functional is the unique minimizer of the expected score. Such scoring functions are called strictly consistent for the functional. The elicitability of a functional opens the possibility to compare competing forecasts and to rank them in terms of their realized scores. In this paper, we explore the notion of elicitability for multi-dimensional functionals and give both necessary and sufficient conditions for strictly consistent scoring functions. We cover the case of functionals with elicitable components, but we also show that one-dimensional functionals that are not elicitable can be a component of a higher order elicitable functional. In the case of the variance, this is a known result. However, an important result of this paper is that spectral risk measures with a spectral measure with finite support are jointly elicitable if one adds the “correct” quantiles. A direct consequence of applied interest is that the pair (Value at Risk, Expected Shortfall) is jointly elicitable under mild conditions that are usually fulfilled in risk management applications.